干细胞向肝细胞定向分化与NF-κB和UBC9表达

发布时间：2018-06-06 21:13

本文选题：原始生殖细胞 + 肝细胞　；参考：《青岛大学》2014年硕士论文

【摘要】：目的：研究小鼠胚胎肝脏发育的过程和体外干细胞向肝细胞定向分化与NF-κB和UBC9表达的关系,探讨干细胞向肝细胞定向分化的分子机制。方法：分离培养扩增小鼠原始生殖嵴细胞(PGCs),向培养液中分别加入20μ/Lβ-神经细胞生长因子(β-NGF)、0.5g/L新生鼠肝脏提取液和胎肝细胞上清液体外诱导小鼠生殖嵴来源的胚胎干细胞(PGCs)向肝样细胞定向分化。观察细胞的形态学的变化；免疫细胞荧光法检测肝样分化细胞内α1-抗胰蛋白酶(AAT)的表达及对NF-κB的表达进行定位；免疫细胞化学法检测肝样分化细胞内UBC9的表达；Real-TimePCR检测在诱导分化过程中NF-κB和UBC9mRNA的相对表达量。免疫组化对胚胎发育过程中11.5d、14.5d、18.5d的肝脏中NF-κB和UBC9的表达进行分析。结果：PGCs培养液中加入20μg/Lβ-NGF、0.5g/L新生鼠肝脏提取液和胎肝细胞上清液可见部分细胞变为圆形或卵圆形；诱导12天后,圆形或卵圆形细胞呈现α1-抗胰蛋白酶阳性表达；在诱导分化过程中NF-κB的表达逐渐增强,伴随肝细胞的成熟而呈阳性表达并由胞质转入胞核。Real-Time PCR结果显示在诱导分化过程中NF-κB的表达量升高,UBC9的表达量增加。而在胎肝发育过程中随肝原细胞向肝成熟细胞转化,肝细胞胞质中NF-κB的表达逐渐增强,并向细胞核内转移；UBC9表达在11.5d-14.5d逐渐增强,18.5d,肝细胞成熟时,UBC9在胞核中几乎不表达。结论：20μg/Lβ-NGF、0.5g/L新生鼠肝脏提取液和胎肝细胞上清液可以诱导小鼠PGCs分化为肝细胞,诱导分化过程中NF-κB的表达逐渐增强,UBC9的表达量升高；而在胎肝发育过程中随肝原细胞向肝成熟细胞转化,NF-κB的表达逐渐增强,UBC9表达先增强后减弱。
[Abstract]:Objective: to study the relationship between the development of mouse embryonic liver and the expression of NF- 魏 B and UBC9, and the relationship between the differentiation of stem cells into hepatocytes in vitro and the expression of NF- 魏 B and UBC9. To explore the molecular mechanism of the differentiation of stem cells into hepatocytes. Methods: the mouse primordial reproductive crest cells (PGCsN) were isolated and expanded, and 20 渭 / L L 尾 -neuronal growth factor (尾 -NGF) and fetal hepatocytes were added to the culture medium, respectively. The supernatant induced the differentiation of mouse embryonic stem cells derived from reproductive crest into hepatocyte-like cells in vitro. The expression of 伪 1-antitrypsin (AATA) and the expression of NF- 魏 B in hepatoid differentiation cells were detected by immunofluorescence assay. Immunocytochemistry was used to detect the expression of UBC9 in hepatoid differentiation cells. Real-time PCR was used to detect the relative expression of NF- 魏 B and UBC9 mRNA in hepatoid differentiation cells. The expression of NF- 魏 B and UBC9 in liver of 11.5d 14.5 d and 18.5d during embryonic development was analyzed by immunohistochemistry. Results some cells became round or oval in 20 渭 g / L 尾 -NGF 0.5 g / L neonatal rat liver extract and fetal liver cell supernatant after 12 days of induction. The positive expression of 伪 1-antitrypsin was observed in round or oval cells, and the expression of NF- 魏 B increased gradually during differentiation. The expression of NF- 魏 B and UBC9 increased during induction and differentiation of hepatocytes from cytoplasm to nucleus. Real-Time PCR results showed that the expression of NF- 魏 B was increased during differentiation. During the development of fetal liver, the expression of NF- 魏 B in the cytoplasm of hepatocytes increased gradually with the transformation of hepatocytes into mature hepatocytes. The expression of UBC9 was gradually increased at 18.5 days after 11.5d-14.5d was transferred into the nucleus, and almost no expression of UBC9 was found in the nucleus of hepatocytes at maturation. Conclusion the liver extract and fetal hepatocyte supernatant of 0.5 g / L of L 尾 -NGFN at 20 渭 g / L can induce the differentiation of 11.5d-14.5d into hepatocytes. During the process of differentiation, the expression of NF- 魏 B increased gradually and the expression of UBC9 increased gradually, while the expression of NF- 魏 B increased at first and then decreased with the transformation of hepatocytes into mature cells during the development of fetal liver.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

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